This invention relates to a class of cyclic peptides that exhibit gram-positive and gram-negative bactericidal activity, as well as fungicidal and anti-mycoplasma activity, and are useful as antimicrobial agents.
The emergence of many medically relevant resistant strains of bacteria is a major issue in human health (Neu, 1992). It is becoming essential that new therapeutic agents be developed to combat microorganisms resistant to traditional antibiotics.
Gramicidin S (GS) is a naturally occurring cyclic peptide antibiotic (cyclo(Val-Orn-Leu-D-Phe-Pro)2) first isolated from Bacillus brevis. There may be a reduced incidence of resistance developed against GS analogs, allegedly because the target of these analogs is the cell membrane of sensitive microorganisms; nonetheless, their mechanism of action is still not well understood. Furthermore, only two proteases are known to degrade GS, GS and its analogs are predicted to be stable in vivo (Maeda, et al., 1993; Yukioka, et al., 1966).
X-ray and NMR studies of GS have confirmed that it forms a two-stranded antiparallel xcex2-sheet structure with the strands fixed in place by two type IIxe2x80x2 xcex2-turns (Izumiya, et al., 1979; Rackovsky and Scheraga, 1980; Hull, et al., 1978). Based on studies of GS and its analogs, a number of structural requirements believed to be important for GS activity have been determined. These include: i) the requirement for an amphipathic structure containing basic residues on the hydrophilic face of the molecule (Izumiya, et al., 1979); ii) a xcex2-sheet structure, or the ability to achieve a xcex2-sheet structure in the presence of lipid bilayers (Izumiya, et al., 1979; Ando, et al., 1993, 1995), and iii) a high overall hydrophobicity (Kondejewski, et al., 1996; Katayama, et al., 1994; Tamaki and Ludescher, 1969).
A drawback to the use of GS as an antibiotic is its ability to lyse certain eukaryotic cells, which can result in a high level of hemolysis. Consequently GS is generally restricted to topical administration.
GS analogs containing more than ten residues have been shown to exhibit a change in activity profile (i.e., a change from gram positive specificity to gram negative specificity) when evaluated using agar-based assays, and to exhibit reduced hemolytic activity compared to GS (Ando, et al., 1993, 1995; Aoyagi, et al., 1988). Further, Aoyagi, et al. (1988) reported on a cyclic 14-mer having activity against gram positive and gram negative bacteria. Tamaki, et al. (1988) present CD data that shows that a 14-mer having two D-Phe residues as a non-L amino acid exists in a beta-sheet conformation, which can be disrupted by incorporation of a third D-amino acid in the peptide.
As mentioned above, despite its desirable properties as a broad spectrum antibiotic, the use of GS is limited by its side effects, specifically, its propensity for hemolysis. Therefore it would be useful to have a broad spectrum, cyclic peptide antibiotic that possess a high level of broad spectrum antimicrobial activity with much reduced hemolytic activity. Peptides having such properties form one aspect of the present invention.
More specifically, it is the discovery of the present invention that cyclic peptides that do not conform to the xcex2-pleated sheet conformation that is characteristic of GS, exhibit superior properties, including broad spectrum antimicrobial activity and a high therapeutic index. The present invention provides guidance for the design and testing of cyclic peptides that exhibit such exceptional properties.
The present invention is generally directed to cyclized peptides that have antimicrobial activity. Such peptides are generally greater than 11 amino acids in length. The invention provides guidance for the design, production, testing and use of such peptides.
In one embodiment, the invention includes an analog of the 14-mer cyclic peptide having the amino acid sequence V1-K2-L3-K4-V5-Y6-P7-L8-K9-V10-K11-L12-Y13-P14 also identified by SEQ ID NO: 1, where Y represents D-tyrosine, numerals represent relative positions in the sequence of the peptide analog, and P14 is linked to V1. The analogs are further characterized by the presence of an amino acid substitution selected from the group consisting of
(i) a D-amino acid in at least one of positions 1-5, 7-12 and 14 or an L-amino acid in position 6 or 13;
(ii) same class substitution at any of positions 1, 3, 5, 8, 10 or 12 with a class I hydrophobic sidechain;
(iii) same class substitution at any of positions 2, 4, 9 or 11 with a class II basic sidechain;
(iv) same class substitution at any of positions 6 or 13 with a D-amino acid comprising a class I hydrophobic sidechain;
(v) transposition of adjacent positions with substitutions of a class I sidechain at any of positions 1, 3, 5, 8, 10 or 12 with a class II basic sidechain and substitution of an adjacent class II sidechain with a class I sidechain to form a non-amphipathic cyclic peptide analog with respect to a plane formed by the peptide backbone of the cyclic structure;
(vi) substitution of class I sidechains for class II side chains at one end of the cyclic structure and substitution of class II sidechains for class I sidechains at the opposite end of the cyclic structure to form a polarized non-amphipathic cyclic peptide having basic residues at one end of the cyclic peptide and hydrophobic residues at the opposite end of the structure; and
(vii) combinations of any of (i)-(vi).
In one embodiment the analog referred to above excludes the peptide known as GS14 (SEQ ID NO: 1). In another embodiment, the analog is further characterized by a disrupted xcex2-sheet structure, as evidenced by a circular dichroism spectrum that is shifted relative to a spectrum measured from a cyclic peptide having the sequence SEQ ID NO: 1 (GS14). In another embodiment, the analog has a specified hydrophobicity, defined in terms of a hydrophobicity window, herein. In a preferred embodiment, the peptide analog is further characterized by a therapeutic index of greater than 1, where the therapeutic index value is determined as a ratio of the concentration of analog required to produce hemolysis of human red blood cells divided by the concentration of analog required to inhibit growth of a specified microbe.
According to further preferred embodiments, the peptide analog is effective against one or more microbes selected from the group consisting of Pseudomonas, Escherischia coli, Salmonella, Staphylococcus, Bacillus, Enterococcus, Corynebacterium, Candida, and mycoplasma.
In another preferred embodiment, amino acid substitution to form the analog comprises substitution of a D-valine at a position selected from the group consisting of position 1 and position 5. In a further preferred embodiment, the amino acid substitution comprises substitution of a D valine at position 10. In still a further embodiment, the amino acid substitution comprises substitution of a D-leucine at a position selected from the group consisting of position 8 and position 12. The amino acid substitution may also include substitution of a D-leucine at a position 3.
In a related embodiment, the peptide analog of the invention has an amino acid substitution of a D-lysine at a position selected from the group consisting of position 2, position 4, position 9 and position 11. A further embodiment includes substitution at any of positions 1, 3, 5, 8, 10 or 12 with an amino acid selected from the group consisting of alanine, valine, leucine, norvaline, isoleucine, norleucine, methionine, phenylalanine, tyrosine and tryptophan. Here, the amino acid substitution is preferably selected from the group consisting of a leucine at each of positions 1, 5 and 10 (V3/L3), an alanine at each of positions 3, 8 and 12 (L3/A3), an alanine at each of positions 1, 5 and 10 (V3/A3), and an alanine at each of positions 1, 3, 5, 8, 10, and 12. Within this group, the amino acid substitution will preferably consist of a leucine at each of positions 1, 5 and 10 and further comprising a D-phenylalanine at each of positions 6 and 13. A further modification of this particular construct can be made by substitution at position 4 by D-lysine.
In a related embodiment, peptides in accordance with the invention are GS14 analogs having amino acid substitutions at any of positions 2, 4, 9 or 11 with an amino acid selected from the group consisting of lysine, arginine, ornithine, histidine, 2,4-diaminobutyric acid and 2,3-diaminoproprionic acid.
In a further embodiment, the peptide analog will have an amino acid substitution at any of positions 6 or 13 with an amino acid selected from the group consisting of a class I hydrophobic D-amino acid. Preferably, such a construct will include a substitution at each of positions 6 and 13 with a D-phenylalanine.
Additional peptide analogs in accordance with the invention include amino acid substitutions that represent a transposition of adjacent positions with substitutions of a class I sidechain at any of positions 1, 3, 5, 8, 10 or 12 with a class II basic sidechain and substitution of an adjacent class II sidechain with a class I sidechain to form a non-amphipathic cyclic peptide analog with respect to a plane formed by the peptide backbone of the cyclic structure. According to this feature of the invention, the substitution may consist of a valine at position 4, a lysine at position 5, a leucine at position 11 and/or a lysine at position 12.
In still another preferred embodiment, peptides of the present invention include peptide analogs of GS14 having a lysine at position 1, a valine at position 2, a lysine at position 3, a leucine at position 4, a lysine at position 5, a lysine at position 8, a valine at position 9, a lysine at position 10, a leucine at position 11, and a lysine at position 12. In a preferred embodiment, such a peptide includes a D-lysine at position 1. In another preferred embodiment, the peptide has a D-valine at position 12.
In another general embodiment, GS14 peptide analogs in accordance with the present invention include substitution of class I sidechains for class II side chains at one end of the cyclic structure and substitution of class II sidechains for class I sidechains at the opposite end of the cyclic structure to form a polarized non-amphipathic cyclic peptide having basic residues at one end of the cyclic peptide and hydrophobic residues at the opposite end of said structure. Such substitutions may be effected by a leucine at position 1, a valine at position 2, a lysine at position 5, a lysine at position 8, a leucine at position 11, and a valine at position 12.
In a related aspect, the invention includes a method of enhancing the therapeutic index of an antimicrobial amphipathic cyclic peptide having a xcex2-pleated sheet structure. According to this aspect of the invention, amino acids in the peptide are replaced so as to to disrupt the xcex2-pleated sheet structure, according to the methods discussed herein. In a preferred embodiment, the amphipathic cyclic peptide has the sequence SEQ ID NO: 1 [GS14] and comprises an amino acid substitution selected from the group consisting of
(i) a D-amino acid at at least one of positions 1-5, 7-12 or 14;
(ii) an L-amino acid in at least one of positions 6 and 13;
(iii) same class substitution at any of positions 1, 3, 5, 8, 10 or 12 with a class I hydrophobic sidechain;
(iv) same class substitution at any of positions 2, 4, 9 or 11 with a class II basic sidechain;
(v) same class substitution at any of positions 6 or 13 with a class I hydrophobic sidechain;
(vi) transposition of adjacent positions with substitutions of a class I sidechain at any of positions 1, 3, 5, 8, 10 or 12 with a class II basic sidechain and substitution of an adjacent class II sidechain with a class I sidechain to form a non-amphipathic cyclic peptide analog with respect to a plane formed by the peptide backbone of the cyclic structure;
(vii) substitution of class I sidechains for class II side chains at one end of the cyclic structure and substitution of class II sidechains for class I sidechains at the opposite end of the cyclic structure to form a polarized non-amphipathic cyclic peptide having basic residues at one end of the cyclic peptide and hydrophobic residues at the opposite end of said structure; and
(viii) combinations of any of (i)-(vii).
In a further preferred embodiment, the method includes making a substitution that is effective to form any of the peptide analogs of claims 2-24.
In a related embodiment, the invention also includes a pharmaceutical composition that includes any of the peptide analogs of claims 1-24, characterized by a therapeutic index of greater than 1 against a specified microbe. Such a composition may also include an acceptable pharmaceutical excipient. Such composition may be effective against gram positive or gram negative bacteria, for example, Pseudomonas, Escherischia coli, Salmonella, Staphylococcus, Bacillus, Enterococcus, or Corynebacterium; against fungi, for example, Candida.
The invention also includes a method of treating a microbial infection in a subject. According to this aspect of the invention, the subject is given any of the peptide analogs of claims 1-24 in an amount effective to produce a microbe-inhibitory concentration of said peptide analog in the subject.
In a related aspect, the invention includes a method of treating a mycoplasma infection or contamination, such as in cell culture. Here, the mycoplasma is exposed to any of the peptides of claims 1-24 in an amount effective to produce a mycoplasma inhibitory concentration of said peptide analog. In a preferred embodiment, the peptide is GS14.
In a more general aspect, the invention includes a cyclic antimicrobial peptide having the sequence [(X1X2)nX1-X3-P], (Examples of these include the sequences presented as SEQ ID NOs: 35-38) where X1 is independently selected from the group consisting of alanine, valine, leucine, norvaline, isoleucine, norleucine, methionine, phenylalanine, tyrosine and tryptophan, X2 is independently selected from the group consisting of lysine, arginine, histidine, ornithine, 2,4-diaminobutyric acid, and 2,3-diaminopropionic acid, X3 is a D-amino acid selected from the class I group of hydrophobic amino acids consisting of of alanine, valine, leucine, norvaline, isoleucine, norleucine, methionine, phenylalanine, tyrosine and tryptophan, and n is an integer that is greater than or equal to 2, said cyclic peptide characterized by a disrupted amphipathic xcex2-pleated sheet structure, as evidenced by a circular dichroism spectrum that is shifted relative to a spectrum measured from a cyclic peptide having the sequence SEQ ID NO: 1 [GS14].
In a preferred embodiment, n is an integer between 2 and 5. In still another preferred embodiment, at least three amino acids comprising said peptide are D-amino acids. More specifically, the peptide has the sequence V1-K2-L3-K4-V5-Y/F6-P7-L8-K9-V10-K11-L12-Y/F13-P14, wherein Y/F designates a D-tyrosine or a D-phenylalanine residue and wherein said third D-amino acid is selected from the group consisting of K2, K4, V5, L8, K9, V10, and K11. In another embodiment, one of positions X1 or X2 is a deletion.
In still another related aspect, the invention includes a cyclic peptide having the sequence (X1X2)n-X 3-P-(X2X1)n-X3-P (Examples of these include the sequences presented as SEQ ID NOs: 39-42), where n is an integer greater than or equal to 2, further characterized as lacking a xcex2-pleated sheet structure, as evidenced by circular dichroism spectrum lacking peaks characteristic of xcex2-pleated sheet structure. Here, each X1 is independently selected from the group consisting of alanine, valine, leucine, norvaline, isoleucine, norleucine, methionine, phenylalanine, tyrosine and tryptophan, each X2 is independently selected from the group consisting of lysine, arginine, histidine, ornithine, 2,4-diaminobutyric acid, and 2,3-diaminoproprionic acid, each X3 is a D-amino acid independently selected from the group consisting of alanine, valine, leucine, norvaline, isoleucine, norleucine, methioine, phenylalanine, tyrosine and tryptophan. In preferred embodiments, this peptide takes the form VKLYYPKVKLYP (GS12, SEQ ID NO:29) LKVKYPKLKVY (GS12LV, SEQ ID NO:30), VKLKFPKVKLFP (GS12F, SEQ ID NO:31), VOLOFPOVOLFP (GS12FO SEQ ID NO:32), or LOLOFPOLOLFP (GS12FO/LL, SEQ ID NO:33). The peptide may also have a deletion at one of the positions X1 or X2.
These and other aspects of the invention can be discerned by persons skilled in the art from the Descriptions and Examples that follow.